The human c-Fes protein-tyrosine kinase is associated with differentiation signaling in myeloid hematopoietic cells, vascular endothelium and neurons. Recent global sequence analysis of tyrosine kinase domains from colon cancer cell lines unexpectedly revealed consistent mutations in the human c-fes gene (Science 300: 949, 2003). While this report speculated that these mutations may induce kinase activation and contribute to tumorigenesis, we found that these mutations suppress kinase activity or have no effect. In addition, Fes expression was readily detected in normal human and mouse colonic epithelium, but was greatly reduced or absent in tumor sections from most colon cancer patients. Fes was also undetectable in five of six colorectal carcinoma cell lines examined, and re-introduction of Fes using a recombinant retrovirus suppressed colony-forming activity in soft agar and invasiveness in the Matrigel assay. These data suggest that c-Fes is a tumor suppressor in colorectal cancer, despite its tyrosine kinase activity. This hypothesis will be tested with the following Specific Aims: Aim 1. Determine the biological impact of c-Fes kinase domain mutations and the mechanism of c- fes promoter silencing in colorectal cancer cell lines and primary tumors. In this Aim, cancer-associated Fes mutants will be tested for loss of kinase activity and growth inhibitory function in colorectal cancer cell-based assays. If c-fes has an important tumor suppressor role in colon cancer etiology, then tumor-associated mutations are predicted to interfere with the ability of Fes to suppress the growth and invasiveness of colon cancer cells lines. In addition, methylation of the c-fes promoter will be examined as a common mechanism of c-fes gene silencing associated with colon tumor development. Aim 2. Test the hypothesis that Fes regulates the differentiation of colonic epithelial cells and that loss of this function contributes to tumorigenesis. This Aim will investigate whether the growth-suppressive actions of Fes observed in colorectal cancer cell lines are linked to differentiation as observed in other cell lineages. Experiments will also address whether Fes is required for differentiation of the Fes-positive colon cancer cell line, Caco-2. Aim 3. Determine whether Fes-knockout mice display enhanced susceptibility to colon cancer development. Mice lacking c-fes or bearing a kinase-inactivating mutation in the c-fes locus will be crossed with ApcMin/+ mice, which spontaneously develop non-invasive intestinal tumors. If c-fes is a true tumor suppressor, then mice lacking c-fes function are expected to develop more severe disease in terms of tumor size, number, and invasiveness.